Background: Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) hinder the progress of TB control.Objective: To track the trend of drug-resistant tuberculosis (DR-TB) prevalence in Zhejiang Province from 1999 to 2013, and identify risk factors of resistance to second-line drugs among MDR-TB patients.Design: Four DR-TB surveys had been done in Zhejiang Province in 1999, 2004, 2008 and 2013 through questionnaires, in which demographic and epidemiological items were included. After questionnaires, drug susceptibility testing (DST) targeted at four first-line drugs was done for all TB patients and DST targeted at six second-line drugs (only in 2008 and 2013) for MDR-TB patients. The drug resistance trend over time was analyzed using the Cochran–Armitage test. The factors associated with resistance to second-line drugs among MDR-TB patients were examined by a multivariate logistic regression model.Results: Of 936 patients enrolled, 27 (3.21%) and 20 (21.28%) MDR-TB cases were registered as new and previously treated cases, respectively. MDR-TB showed a decreasing trend  while resistance to any first-line drugs showed an increasing trend , from 1999 to 2013. The highest resistance rate was shown to ofloxacin among MDR-TB patients both in 2008 (28.8%) and in 2013 (27.7%), while resistance to para-aminosalicylate decreased significantly  between 2008 and 2013. MDR-TB patients aged 45–65 years  were more likely to be resistant to any second-line drugs.Conclusions: DR-TB including MDR-TB remains a major public health problem in Zhejiang Province. Further efforts on MDR-TB control should be conducted to hinder drug resistance, including critical clinical use of anti-TB antibiotics and preventing transmission. The prevalence of multidrug-resistant tuberculosis (MDR-TB) in China was estimated to be 1.7 and 1.3 times of the global average among new and previously treated patients respectively in 2014 ,6. HowevZhejiang Province is one of the most developed provinces in China and has constructed a routine monitoring system for drug resistance since the World Health Organization (WHO) integrated it into its surveillance network of DR-TB in 1999. According to the code of the WHO, Zhejiang conducted four cross-sectional surveys on drug resistance in 1999, 2004, 2008 and 2013. DST for first-line drugs  was implemented in each survey but DST for six second-line drugs  among MDR-TB was only available in 2008 and 2013.Our previous study showed that the prevalence of MDR-TB decreased from 8.6% in 1999 to 6.0% in 2008 and INH resistance was related to RFP resistance [20.050.5(1 − 0.5)/(0.07/2)2 = 784) to achieve 95% precision and a margin of error of 7%, assuming no prior knowledge of prevalence of drug resistance, to measure prevalence of drug resistance across Zhejiang Province in each survey. Assuming that > 10% of samples would be lost, we sought to obtain 900 cases. We randomly selected 30 TB treatment centers in 30 counties among 90 centers in Zhejiang Province and planned to recruit 30 sputum smear-positive patients in each center. Details can be found in our previous study (Jia) [Our study aimed to evaluate levels and trends of DR-TB in Zhejiang Province. All 30 counties surveyed in 1999 were included in the fourth survey in 2013 except for one county. We would need 784 cases  smear microscopy and solid LJ culture were used. The identification of eviously .p-values < 0.05 as significant, and < 0.001 as highly significant. The Cochran–Armitage trend test and logistic regression were done using SAS 9.1 . Multilevel multivariate logistic regression analysis was done with MLwiN 2.02 (Multilevel Models Project Institute of Education).The drug resistance trend over time was analyzed using the Cochran–Armitage test for trend. The factors associated with MDR-TB were examined by a multilevel multivariate logistic regression model, in which the region was fitted as 3-level to detect spatial aggregation, year of survey as 2-level and each individual as 1-level. A multivariate logistic regression model was used to identify independent factors associated with resistance to any second-line drugs among MDR-TB patients, by estimating OR and 95% CI for each factor. We took A total of 1010 sputum-positive patients were included in the fourth TB drug resistance surveillance of Zhejiang Province. The survey was performed from February 2013 to August 2014. Of these, 23 smear samples showed negative results  and 51 strains were detected as non-TB mycobacterium. The final study population consisted of 936 individuals.p < 0.001).There were no statistically significant demographic differences between non-MDR-TB and MDR-TB patients . PatientFactors associated with resistance to MDR-TB are shown in p < 0.01) and an increase (from 20.3% to 30.9%) of proportion resistant to any first-line drugs  in the period of 1999–2013. However, the proportions remained steady from 2008 to 2013 for both MDR-TB  and resistance to any first-line drugs . For new TB patients, proportion of MDR-TB declined consistently without significance , while proportion of resistance to any first-line drugs increased sharply from 14.15% in 1999 to 29.22% in 2013 . By contrast, those figures for previously treated patients fluctuated over the span of 15 years. In 2013, 29.2% of new patients and 45.7% of previously treated patients were resistant to at least one first-line drug. The proportions of resistance to EMB and SM in 1999–2013 are described in Table S1.p = 0.04) from 2008 to 2013. Of all the six second-line drugs, OFX showed the highest resistance rate of 28.8% in 2008 and 27.7% in 2013. The lowest-resistance drug was identified as CS (5.8%) in 2008, and AM (4.3%) and CPM (4.3%) in 2013. Factors associated with resistance to second-line drugs are shown in p < 0.05). Previously treated patients were not at a higher risk of being resistant to any second-line drugs compared with new patients . Factors like sex, occupation and year were also not associated with resistance to any second-line drugs.A total of 52 and 47 MDR strains were tested for susceptibility to six second-line drugs in 2008 and 2013 respectively. Of these, 27 (51.9%) and 17 (36.2%) strains showed resistance to at least one second-line drug with no significant difference . The ratThe primary finding in our updated analysis presented an insignificant decrease of proportion of MDR-TB from 6.0% to 5.0% between 2008 and 2013 in Zhejiang, as well as in both new cases (3.3% to 3.2%) and previously treated cases (29.1% to 21.3%). The decline  in 2008–2013 was slightly slower than that in 1999–2004  and in 2004–2008 . This result was mainly attributable to increased resistance to SM, which had been used for many years as the first antibiotic for TB treatment . HoweverPrevious TB treatment was not surprisingly a predictor for MDR-TB, which was the same case in previous studies –18. HoweResistance to six second-line drugs has been tested in 2013 and unoptimistic results have been disclosed. Five of the six second-line drugs did not present a significant decline between 2008 and 2013. Resistance to OFX among MDR-TB patients ranked second in 2008 (28.9%) and first in 2013 (27.7%), with figures higher than those of the Chinese national average 25.4%), Thailand (15.0%), South Africa (4.8%) and India (9.0%) . OFX is .4%, ThaiThe patients aged 45–65 had a higher risk of resistance to second-line drugs  compared with patients under 25. This may be attributable to the side effects of first-line drugs and the economic status of this population. The first-line drugs were reported to be age-related with hepatotoxicity, in addition the second-line drugs are not free for TB treatment in China because of high cost ,27. PatiM. tuberculosis strains. Gao Qian’s study reported that most Chinese DR-TB patients were primary cases [We did not find a higher risk of resistance to second-line drugs among the previously treated patients, which was not in accordance with some studies . Resistary cases .The current study has limitations. First, we could not detect all potential risk factors because of limited resources and non-availability of information. Potential risk factors like HIV status, diabetes, living conditions and income were not measured. This might have led to inaccurate estimation of their true effects. Further, the surveillance only represented patients diagnosed in CDCs and hospitals; private health sectors were not included. Those not having access to health facilities were also not included in our study, therefore there was bias when it came to representing the community’s situation. Nevertheless, given the little information available about second-line drugs among MDR-TB in China, our work can provide implications for MDR-TB control in similar contexts, and generally give implications of the trend of the TB resistance rate in Zhejiang over a period of 15 years.M. tuberculosis strains on the prevalence of DR-TB are considerable issues for future studies.Our findings from monitoring the DR-TB trend against both first- and second-line drugs in Zhejiang Province provided general implications on TB control to regions with similar contexts. The impact of general clinical use of anti-TB antibiotics and transmission of drug-resistant


Background. Our aim was to evaluate the protective effect of lipoic acid (LA) on fetal outcome of diabetic mothers. Methods. Diabetes was induced in female rats using streptozotocin and rats were made pregnant. Pregnant control  or pregnant diabetic  rats were treated daily with either LA (groups 2 and 4) or vehicle (groups 1 and 3) between gestational days 0 and 15. On day 15 of gestation, the fetuses, placentas, and membranes were dissected, examined morphologically, and then homogenized, to measure cyclooxygenase (COX) activities and metabolisms of prostaglandin (PG) E2 (PGEM) and PGF2α (PGFM) levels. The level of total glutathione was measured in the maternal liver and plasma and in all fetuses. Results. Supplementation of diabetic rats with LA was found to significantly (p < 0.05) reduce resorption rates in diabetic rats and led to a significant (p < 0.05) increase in liver, plasma, and fetuses total glutathione from LA-TD rats as compared to those from V-TD. Decreased levels of PGEM and elevated levels of PGFM in the fetuses, placentas, and membranes were characteristic of experimental diabetic gestation associated with malformation. The levels of PGEM in malformed fetuses from LA-TD mothers was significantly (p < 0.05) higher than those in malformed fetuses from V-TD rats. Conclusions. LA treatment did not completely prevent the occurrence of malformations. Thus, other factors may be involved in the pathogenesis of the diabetes-induced congenital malformations. These congenital malformations most commonly involve the central nervous, cardiovascular, and skeletal systems . There h2 (PGE2) , 5. Furt2 (PGE2) –11. Incr glucose . The add glucose . In vivoitamin E , vitaminitamin E , and lipα-LA was shown to reverse apoptosis, ameliorate mitochondrial deformation, and increase the amount of mitochondrial DNA [Lipoic acid  is found inside every cell of the body. Lipoic acid is a potent antioxidant, capable of inducing its activity in both a lipophilic and an aqueous milieu , 13. It rial DNA .A healthy body makes enough LA to supply its energy requirements; therefore, there is no daily requirement for this supplement. However, several medical conditions appear to be accompanied by low levels of LA—specifically, diabetes, liver cirrhosis, and heart disease —which su2 and F2α metabolites in diabetic rats.At present, the exact mode of action of LA has not been fully elucidated. This research study is designed to investigate the protective nature of LA on fetal outcome while determining the processes involved in diabetic fetopathy. To achieve this, the study will examine the effects of LA on the activity of cyclooxygenases and the levels of prostaglandins EThis study will make major contribution to our nation as it will reduce the rate of congenital malformation and will be of considerable benefit to health sector since congenital malformation imposes a substantial cost to the health budget and emotional burdens upon society.All animal studies were reviewed and approved by the Animal Ethics Committee of University of Petra (no. ANIM: LAGES-2014-04). The guide for the care and use of laboratory animals was followed. ad libitum.Nulliparous female rats from a local Wistar-derived strain, 60 to 70 days of age with initial weights of 220 ± 30 g, were used. They were housed in rooms maintained at a temperature of 21°C ± 3°C and 12 : 12 h light/dark cycle. A commercial diet and tap water were providedn = 10) and 4 (n = 8) by a single intraperitoneal (IP) injection of freshly prepared streptozotocin , while control animals groups 1 (n = 9) and 2 (n = 7) were injected with a buffer solution . One week after the injections, venous tail blood was collected and hyperglycemia was confirmed with blood glucose levels greater than 11 mmol/L using a glucometer.The rats were randomly distributed to four groups, two control groups  and two diabetic groups (groups 3 and 4). Diabetes was induced in female rats from groups 3 (The rats (both controls and diabetic) were mated with healthy nondiabetic males overnight. The presence of a sperm mucus plug in the vagina the following morning signified pregnancy. Further, the presence of spermatozoa was designated as day zero of pregnancy. Pregnancy was confirmed by subsequent increases in body weight.Thereafter, rats were daily injected IP with either LA  or vehicle only (groups 1 and 3) between gestational days 0 and 15.At day 15 of gestation, pregnant diabetic and control rats were sacrificed by cervical dislocation, and maternal blood samples were collected by cardiac puncture. In each sacrificed rat, the uterus was exposed by cesarean section, and the number of implantation and resorption processes was recorded. The live fetuses, along with their placentas and membranes, were dissected out of the uterine horns, rinsed carefully in phosphate buffered saline (PBS), and carefully labeled. Overall growth and differentiation of the fetuses were quantified by direct measurement of crown-to-rump length (CRL). The fetuses were examined for their general morphology, and the presence of any disturbed fetal development, such as an open neural tube or growth retardation, and the appearance of specific parts and organs  were also noted. This examination was conducted by a researcher masked to the various experimental groups. Fetuses with apparent anomalies in one or more of these structures were considered “malformed.” Fetal, placental, and membrane weights were also recorded. The fetuses, placentas, and membranes were then snap-frozen in liquid nitrogen and kept at −80°C for subsequent analysis. in vitro by the addition of supernatants from the homogenates of placentas, which could have contained such an inhibitor, to the incubated media, and found that such activity was not affected.To rule out the possible presence of COX-1 inhibitor in association with pregnancy, we examined the activity of COX-1The COX activity assays were determined according to procedures used previously . Protein2α and PGE2 are rapidly converted in vivo to their 13,14-dihydro-15-keto metabolites PGFM and PGEM, respectively, [2α and PGE2.Circulating prostaglandin (PG) PGFctively, , 21 withPGFM and PGEM were measured by enzyme immunoassays (EIA)  with a sBiochemical parameters were determined for all individual fetuses and placentas, while membranes from each group were pooled to allow determination of all parameters in the same sample. PGs metabolites were also assayed in maternal blood.μmol/g tissue and μmol, respectively. The level of total glutathione was measured also in the fetuses of all groups and expressed as μmol/mg of protein. The kits for COX, EIA for both PGFM and PGEM, and total glutathione levels were from the Cayman Chemical Company .Liver samples from the maternal rats were removed, then snap-frozen in liquid nitrogen, and kept at −80°C for subsequent analysis. Liver and plasma total glutathione levels, a sensitive indicator of oxidative stress, were determined and expressed as  p value was smaller than 0.05. Data are presented as means ± SEM.Statistical analysis of the present study data was performed by a biostatistician using Shapiro-Wilk, Kruskal-Wallis, Tukey, and/or Mann-Whitney one-way analysis of variance rank sum tests . Statistical difference for overall malformation between both diabetic groups (vehicle-treated diabetic and LA-treated diabetic) was calculated using a chi-squared test. Results were considered statistically significant when thep < 0.05) (five times) than comparable samples obtained from both groups of control pregnant rats (The average glucose levels in random blood samples from diabetic pregnant rats were significantly higher (ant rats .p < 0.05) than samples obtained from both groups of control rats. Daily injections of LA did not normalize blood glucose levels of diabetic rats to the control rats and did not significantly affect the blood sugar concentrations of both diabetic and control rats.Furthermore, the average glucose levels in samples obtained from LA-treated diabetic (LA-TD) rats were also significantly higher  resorption rates from 5.80 ± 2.15 in V-TD rats to zero in LA-TD rats.In diabetic rats, LA supplementation significantly reduced (p < 0.05) in comparison to both groups of control and LA-TD rats.Fetal body mean weight in V-TD rats was significantly decreased (p < 0.05) than those in V-TC rats. In contrast, membrane mean weight in both groups of control rats was significantly higher (p < 0.05) than those in both diabetic rat groups.Placental mean weight from both groups of diabetic rats was significantly higher  in mean CRL was observed, in comparison with offspring of V- and LA-TC or LA-TD rats. Supplementation of diabetic rats with LA increased CRL in their offspring and was able to normalize the measure to those in V- and LA-TC rats.To estimate embryonic development, we measured fetal crown-rump length CRL, . In offsp = 0.06).Administration of LA to diabetic rats for 15 days during gestation marginally reduced the occurrence of malformations . The incIn the V-TD rats, 41.66% of fetuses had growth retardation, as represented by CRL, 39.58% had short mandibles, 31.25% had open neural tubes, 12.5% had malrotations, and 2.08% had short tails. However, in LA-TD rats, the corresponding proportions were 15.25%, 10.17%, 13.56%, 5.09%, and 0.0%, respectively. Some fetuses exhibited a single malformation, while others had multiple anomalies.To assess the ability of LA to reduce the maternal oxidative stress associated with diabetes, we measured the level of total glutathione in the maternal liver and plasma.p < 0.05) glutathione content in comparison with those in V- and LA-TC rats.As seen in p < 0.05) in liver total glutathione in comparison with V-TD rats, although such supplementation failed to normalize glutathione content to those in the liver of both groups of control rats. Furthermore, LA treatment significantly increased (p < 0.05) the content of total glutathione in the plasma of diabetic rats compared with those in V-TD rats. This value was not found to be significantly different from that observed in both groups of control rats.Supplementation of diabetic rats with LA leads to a significant increase .p < 0.05) than those in both control groups.The total glutathione contents in fetuses of V- and LA-TD rats were 51.17% and 45.17% less than in those of V- and LA-TC rats, respectively. On the other hand, the contents of total glutathione in fetuses of both diabetic rats were significantly lower (p < 0.05) total glutathione content of their fetuses as compared with fetuses from V-TD rats.LA supplementation to diabetic rats significantly increased  compared with fetuses from V-TC, LA-TC, nonmalformed V-TD, and nonmalformed LA-TD mothers. Statistically, there was no significant difference between both control and nonmalformed diabetic fetuses.The activity of COX-1 showed no significant variations among fetuses from all groups . Howeverp < 0.05) of COX-1 activity was associated with placentas from malformed V-TD and LA-TD fetuses compared with placentas of nonmalformed fetuses  than nonmalformed fetuses.A similar trend was found in activities of COX-2; the activities of COX-2 in V-TD and LA-TD placentas from malformed fetuses were significantly lower (p < 0.05) compared with those from both nonmalformed and malformed fetuses of diabetic mothers.The activities of COX-2 in the placentas from both control fetuses were significantly higher (p < 0.05) in the activities of COX-2 in the placentas from malformed fetuses in comparison with those from V-TD rats. Thus, LA treatment to diabetic mothers failed to normalize activities of COX-2 to the control rats.Supplementation of diabetic rats with LA leads to a significant increase (p < 0.05) in comparison to membranes from both the control fetuses (p < 0.05) compared with the nonmalformed fetuses of V-TD mothers. The activities of COX-1 in membranes showed no significant variations among fetuses from all groups.The total activity of COX in membranes from the both diabetic was significantly lower ( fetuses . Furtherp < 0.05) than those in the membranes of fetuses from V-TC, LA-TC, and nonmalformed fetuses from V-TC and LA-TD mothers. Furthermore, the activity of COX-2 in membranes from nonmalformed fetuses of LA-TD was significantly higher (p < 0.05) than those in nonmalformed fetuses from V-TD rats.The activities of COX-2 in membranes of malformed fetuses from V-TD and LA-TD were significantly lower  than those in both control groups.As shown in p < 0.05) than in the malformed fetuses from V-TD and LA-TD mothers, although the levels of PGEM in malformed fetuses from LA-TD mothers were significantly higher (p < 0.05) than those in malformed fetuses from V-TD rats. LA treatment to diabetic mothers failed to normalize levels of PGEM to that of both groups of control rats.The mean value for PGEM in nonmalformed fetuses was significantly higher (p < 0.05) than those in nonmalformed V-TD and LA-TD mothers.In contrast to PGEM, the greatest increases in PGFM were found in fetuses from malformed V-TD and LA-TD mothers and were significantly higher (p < 0.05) than that in V-TC and LA-TC.The mean concentrations in malformed fetuses of both diabetic groups were also significantly higher (p < 0.05) than the mean values in control groups.In placentas from both groups of diabetic fetuses, PGEM levels were significantly lower (p < 0.05).The decrease in placenta PGEM associated with malformed fetuses was even more precipitous in both V-TD and LA-TD and below than that in the control groups and those of nonmalformed samples of both diabetic fetuses (p < 0.05) than those from nonmalformed fetuses of both diabetic and control groups. The average levels in placentas from nonmalformed V-TD and LA-TD fetuses were significantly higher (p < 0.05) than in V-TC and LA-TC fetuses.PGFM was also raised in placentas of both diabetic groups in a pattern similar to that in fetuses from both groups of diabetic mothers . The incAlthough the average levels in placentas from malformed (V-TD and LA-TD) fetuses were higher than those from placentas without malformation, this difference was not statistically significant.p < 0.05) than those in the controls.PGEM levels in the membranes of both diabetic fetuses were significantly lower (p < 0.05) than those in the V-TC and LA-TC.In the membranes of nonmalformed V-TD and LA-TD fetuses, the mean levels of PGEM were significantly lower (p < 0.05) those of nonmalformed fetuses of both diabetic groups. Further, supplementation of diabetic mothers with LA significantly increased (p < 0.05) levels of PGEM in membranes of fetuses  compared with those in the V-TD fetuses.The mean levels of PGEM in the membranes of malformed V-TD and LA-TD fetuses were significantly below (p < 0.05) than those in the control groups.The smallest increases in PGFM were observed in fetal membranes . The levp < 0.05) than those in the V-TC and LA-TC.The mean PGFM levels in membranes from V-TD nonmalformed and malformed fetuses were significantly higher (p < 0.05) than those in the controls. Furthermore, the increases in mean PGFM levels in LA-TD  were not statistically significant compared with those in V-TD mothers.In a pattern similar to that found in membranes from V-TD fetuses, mean PGFM levels in membranes from LA-TD nonmalformed and malformed fetuses were significantly higher (p < 0.05) than those in both diabetic mothers. Furthermore, mean PGEM plasma levels in the controls were also significantly higher (p < 0.05) than mean PGFM levels in both controls.The mean PGEM plasma levels in the controls were significantly higher (p < 0.05) in the mean PGFM plasma levels of V-TD and LA-TD compared with controls  than those from V-TD rats. In contrast to the PGEM, the mean PGFM levels in V-TD mothers plasma were significantly higher (p < 0.05) than those in LA-TD mothers.The plasma levels of PGEM in LA-TD mothers were significantly higher  as opposed to the ones that were not treated (66.66%). Thus, the present study is in agreement with the findings of Sugimura et al. , who conγ-GCS), thereby reducing GSH concentration [Diabetes further weakens the antioxidant system, especially the enzyme for glutathione (GSH) synthesis and gamma-glutamylcysteine ligase  of V-TD and LA-TD mothers was significantly lower compared with that in fetuses from both the controls and nonmalformed V-TD and LA-TD mothers. Thus, the present study is in line with others' findings , which rp < 0.05) than those in nonmalformed fetuses from V-TD rats. This may suggest a protective effect of LA on fetal outcome.Furthermore, the activity of COX-2 in membranes from nonmalformed fetuses of LA-TD was significantly higher  system, and it is a required cofactor for PGE isomerase.A functional decrease in the amount of prostaglandins may be associated with oxidative stress. Whorton et al.  showed t2 production rather than PGF2α as the conversion of PGH2 to PGE2 is 150-fold greater than the conversion of PGH2 to PGF2α. The data from the present study for levels of PGEM and PGFM are not in concordance with those reports. The results from this study and another study [Previous studies by Madore et al.  and Aroser study  showed tFurthermore, the fetuses, placentas, and membranes from LA-TD mothers demonstrated relatively higher PGEM levels. This further demonstrates that LA plays a very important role in trying to maintain PGEM and PGFM levels within normal levels in the body.2α in humans is derived from the isoprostane pathways, which are PG-like compounds formed in vivo from the free radical-catalysed peroxidation of arachidonic acid without the activity of COX enzyme, an alternative route for the formation of PGF2α in vivo.The elevated levels of PGFM in the malformed fetuses of both diabetic groups reported in the present study may reflect oxidative stress status, as has been suggested by El-Bassiouni et al. . Yin et 2α in diabetic gestation. This dogma has further support from the Stephens et al. [Prostaglandin metabolism in rats follows the same route as in humans and is different from those in mice and rabbits . Consides et al.  study, wThis research therefore determines that administration of LA to diabetic pregnant mothers can protect against weight loss and promote the fetal growth developments. Furthermore, diabetes and thus malformation are associated with lower total glutathione contents. LA supplementation to diabetic mothers restored maternal liver total glutathione content to that of control groups.LA treatment to diabetic mothers failed to normalize activities of COX-2 in fetuses and placentas to those in control rats and increased levels of PGEM in malformed fetuses as compared to V-TD but failed to normalize it to levels of both groups of control rats. In contrast to PGEM, the greatest increases in PGFM were found in malformed fetuses from V-TD and LA-TD mothers. Furthermore, LA supplementation increased plasma levels of PGEM than those found in V-TD rats.Since LA treatment did not completely prevent the occurrence of malformations, other factors may be involved in the pathogenesis of diabetes-induced congenital malformations. Hence, the possibility of combining LA with other effective agents such as vitamin E or folic acid to prevent malformations in fetuses of mothers with diabetes is interesting.


We found differential expression of amyloid and tau across the disease groups, and across hippocampal sub-regions. Among amyloid markers, the pattern of Aβ-42 immunoreactivity was similar to that of total Aβ. Tau immunoreactivity showed highest expression in the AD and mixed AD and vascular dementia, AD_VaD, which was higher than in control, post - stroke and VaD groups (p < 0.05). APOE ε4 allele positivity was associated with higher expression of amyloid and tau pathology in the subiculum and entorhinal cortex of post-stroke cases (p < 0.05). Comparison between PSND and PSD revealed higher total Aβ immunoreactivity in PSND compared to PSD in the CA1, subiculum and entorhinal cortex (p < 0.05) but no differences between PSND and PSD in Aβ-42, Aβ-40, soluble Aβ or tau immunoreactivities (p > 0.05). Correlation of MMSE and CAMCOG scores with AD pathological measures showed lack of correlation with amyloid species although tau immunoreactivity demonstrated correlation with memory scores (p < 0.05). Our findings suggest hippocampal AD pathology does not necessarily differ between demented and non-demented post-stroke subjects. The dissociation of cognitive performance with hippocampal AD pathological burden suggests more dominant roles for non-Alzheimer neurodegenerative and / or other non-neurodegenerative substrates for dementia following stroke.Neuroimaging evidence from older stroke survivors in Nigeria and Northeast England showed medial temporal lobe atrophy (MTLA) to be independently associated with post-stroke cognitive impairment and dementia. Given the hypothesis ascribing MTLA to neurodegenerative processes, we assessed Alzheimer pathology in the hippocampal formation and entorhinal cortex of autopsied brains from of post-stroke demented and non-demented subjects in comparison with controls and other dementias. We quantified markers of amyloid β  and hyperphosphorylated tau in the hippocampal formation and entorhinal cortex of 94 subjects consisting of normal controls ( Stroke accounts for half of the global burden of neurological disorders while remaining the most common cause of acquired disability, and a common cause of cognitive impairment and dementia  compared to controls although there was a trend of increased deposition of Aβ-42 over Aβ-40   at the Campus for Ageing and Vitality, Newcastle University, UK. Table Autopsies were performed between 24 and 92 h after death and brains were fixed for between 6 and 34 weeks. Cognitive scores on the Mini-Mental State Examination (MMSE) and Cambridge Cognitive Examination (CAMCOG) proximate to death as well as APOE genotypes were retrieved from clinical and research records of the subjects in the CogFAST-Newcastle cohort. The CogFAST-Newcastle Study and ancillary studies had ethical approval from the local Newcastle Ethical committees and participants gave written consent for brain tissue donation. Use of brain tissue was also approved by the local Ethical committees  and the committee of the NBTR.Post-mortem reports were retrieved for all the cases used in this study. Primary neuropathological diagnoses were made from brain tissue sampled at several coronal levels  as shown in Table Paraffin embedded brain tissue blocks taken from relevant coronal levels of the Newcastle Brain Map . Five images were taken at random from the CA1, CA3 and subiculum, 3 images from CA2 and 4 × 3 from the EC from the pial surface to the white matter. Approximately 2,820 images were taken.Images were analyzed using Image Pro Plus 6.3 . The area of interest (AOI) was delineated on-screen using the wand tool and the size of the area selected was recorded. Histogram based analysis was used to measure the number of pixels stained  and intensity of stain , as determined by the operator. Background was excluded by highlighting only the positive staining. To standardize the measurements, the range of stain intensity for each AOI was determined between 0 and 255 . Using the sum of each of the three measures, further calculations were undertaken to generate the metrics of immunostaining: Percent of the area of interest positively stained, “Percent area (%PA)” = Per Area × 100; Mean intensity of stain per pixel, “Integrated Optical Density (IOD)” = 255 – (sum IOD/area). The mean % PA and IOD were then calculated for each subject by computing averages from the images taken from each hippocampal sub-region.post-hoc analysis for between-group differences) and non-parametric tests Kruskal–Wallis and Mann–Whitney U-tests) for non-normally distributed dataset. The relationship among markers, and with demographic, cognitive and pathological variables were assessed using Pearson's correlation coefficient (r) or Spearman's correlation (rho) as necessary depending on the normality of the dataset. Appropriate power calculation was performed using the G*Power software . The Kolmogorov-Smirnov Test was used to establish normality of data. Comparisons across groups of cases and across sub-regions were performed using parametric tests (ANOVA for group means and Tukey p = 0.786), gender distribution (p = 0.493), post-mortem delay (p = 0.902) and length of fixation of tissues (p = 0.589) across the groups. However, the PSND group had significantly higher scores on the cognitive batteries MMSE and CAMCOG compared to the PSD group (p < 0.05). Similarly, Braak stage, CERAD score and Thal stage were significantly higher in the AD, AD-VaD groups compared to the VaD and post-stroke groups (PSND and PSD) (p < 0.05). Hippocampal vascular scores were similar among the PSND, PSD, VaD and AD-VaD groups. The distribution of the ε3 and ε4 alleles of APOE were not significantly different between the PSND and PSD groups . Given a total sample size of 94, a significance level, α = 0.05 and assuming a moderate effect size Cohen's d = 0.4, 6 sub-groups and 5 degrees of freedom, the computed Power (1-β) = 0.8424 using the G*Power software , demented (PSD) subjects from the CogFAST-Newcastle study; Control, VaD, AD and mixed AD-VaD groups] are shown in Table In quantifying amyloid burden both parenchymal as well as vascular amyloid immunoreactivities were incorporated in order to capture the total quantity of the different species of amyloid detected within the defined area of interest as previously performed  measures in the hippocampal sub-regions and entorhinal cortex. Furthermore, there was significant positive correlation between scores on the semi-quantitative amyloid rating scales of CERAD, Thal, Braak and tau stages with the metrics of 4G8 total IR in the entorhinal cortex, subiculum and CA1 sub-regions Table  suggestip = 0.008, Kruskal-Wallis Test) , showed a trend with AD-VaD (p = 0.064) and PSND (p = 0.076) but not significantly different between PSD and VaD. In the CA2 and CA3 regions, there was no significant variation across disease groups. In the subiculum, there was significant variation across disease groups and controls  with total IR being significantly higher in the AD (p = 0.001), AD-VaD (p = 0.008) and PSND (p = 0.015) groups but not significantly different between PSD and VaD. In the entorhinal cortex, total Aβ IR similarly showed significant variation across the sub-region  with values significantly higher in AD (p = 0.022), AD-VaD (p = 0.016) than in controls while PSD was significantly lower than PSND (p = 0.019), AD-VaD (p = 0.002) and AD (p = 0.002)   and AD-VaD (p = 0.005), but not significantly different from VaD, PSND and PSD  with the IR being significantly higher in AD-VaD (p = 0.046) and VaD (p = 0.014) groups compared to the control and PSD groups respectively . Compared to the PSND group, immunoreactivity was significantly higher in AD (p < 0.001) and AD-VaD (p = 0.002) groups but not significantly different from control PSD and VaD groups  and was significantly higher in the AD (p = 0.001) and AD-VaD (p = 0.008) groups compared to the control and VaD groups respectively. However, the VaD groups did not differ significantly from the PSND and PSD groups respectively  and was significantly higher in the AD (p = 0.001) and AD-VaD (p = 0.001) groups but not different from the VaD, PSND and PSD groups.In the CA1 region, ) Figure . BetweenD Figure . In the  Figures , 5. In ts Figure . In the p = 0.001, Kruskal–Wallis Test). In CA1, the IR in the control group was not significantly different from that of PSND, PSD and, VaD but was significantly lower than AD (p = 0.004) and AD-VaD (p = 0.010). There was no difference between the PSND and PSD groups. In CA2, the control group IR was lower than AD, VaD and AD-VaD although this did not attain statistical significance (p > 0.05). However, VaD (p = 0.005) and AD-VaD (p = 0.006) groups were significantly higher than the PSD group. The PSND group did not differ from the PSD group . There was no significant variation in the CA3 region and subiculum.Immunostaining and distribution of T-40 antibody across disease groups varied significantly across the sub-regions .p > 0.05, Kruskal Wallis Test). However, in the subiculum and entorhinal cortex, the immunoreactivity varied significantly . Between group analysis showed significantly higher immunoreactivity in the AD-VaD (p = 0.007 Mann–Whitney U) group compared to the control group, and in the AD-VaD (p = 0.008) and AD (p = 0.040) compared to the PSD group . There was slightly higher AT8 immunoreactivity in PSD compared to the PSND group in the CA2 and CA3 sub-regions   in the post-stroke cohort, the immunoreactivity being significantly higher in APOE ε4 positive subjects in the subiculum   were APO ε4 positive compared to 3 out of 13 PSD cases (23.1%). Overall, 11 out of 29 (37.9%) post-stroke subjects were ) Figure . Presenc) Figure .p = 0.024), whereas there was no significant correlation with Aβ-42 immunoreactivity.We explored relationships between the various markers of Alzheimer pathology used in this study and cognitive scores of the post-stroke group utilizing Spearman correlation analysis. Measures of general cognitive functioning  and functioning in the memory domain (CAMCOG memory) were correlated with two measures of Alzheimer pathological burden: Aβ-42 immunoreactivity (being the predominant β-amyloid species deposited in the hippocampus) and tau immunoreactivity measures in the CA1, subiculum and entorhinal cortex: sub-regions which demonstrated the most consistent patterns of variation of immunoreactivity across the hippocampal formation. Table APOE ε4 allele positivity and higher load of amyloid and tau pathology in the subiculum and entorhinal cortex of post-stroke cases (4) hyperphosphorylated tau immunoreactivity did not differ significantly between PSND and PSD groups and (5) poor correlation of cognitive measures with the burden of amyloid and tau pathology in post-stroke subjects.We hypothesized that Alzheimer pathology would be differentially expressed in demented and non-demented post-stroke subjects in comparison to normal aging controls and other dementias. We found (1) amyloid beta deposition was not remarkably different between PSD and PSND groups despite the finding that MTLA was a significant feature in PSD subjects. (2) As expected, concentrations of total amyloid beta and amyloid β-42 were significantly greater in AD and mixed subjects but low in post-stroke subjects consistent with insufficient concentrations for a diagnosis of AD (3) an association between Consistently across all the markers of amyloid pathology, we found evidence of increasing accumulation of amyloid in controls, post-stroke groups, AD and AD_VaD in that order. Our finding of amyloid accumulation in normal aging controls concurs with the biological phenomenon of aging-associated accumulation of amyloid that has been reported across species: in drosophilia  of which pyroglutamate-modified Aβ peptides are predominant components. Most N-truncated Aβ are considered to be the degradation products of full-length Aβ although Aβ11–40/42 may be generated intracellularly directly from APP by BACE proteolysis occurring in trans-Golgi network  of the PSND subjects. Despite the limited size of the sub-cohort, the amyloid load was significantly higher in the PSND group than the PSD group. APOE ε4 has been associated with accumulation of amyloid and/or tau pathology  as well as higher Aβ-(42) levels (Jones et al., APOE ε4 allele might have contributed to the higher quantity of amyloid in subregions of the PSND group compared to the PSD group, but further research is required to explore this relationship further.Further analysis of the post-stroke cohort suggested that the presence of the In the post-stroke cohort with available cognitive scores, very limited correlation of tau pathology with CAMCOG memory score was established. In this cohort, there was dissociation of cognitive performance and hippocampal Alzheimer pathologic burden. The implication of this may be that AD pathology probably does not contribute very strongly to the substrates of dementia after a stroke event as previously hypothesized (Henon et al., In conclusion, we did not find quantitative AD pathological markers sufficient to separate the non-demented post-stroke from demented post-stroke subjects. Thus, hippocampal AD-pathological mechanisms do not separate non-demented from demented stroke subjects. Neocortical Alzheimer pathology, other non-Alzheimer neurodegenerative pathologies as well as other non-neurodegenerative mechanisms such as vascular and inflammatory/immune mechanisms require further research in order to fully determine the precise pathological substrates of dementia following stroke.ROA performed experiments in the study and drafted the first draft of the manuscript. AO provided technical support. LA and AO contributed to critically revising the manuscript for important intellectual content. RK corrected drafts and obtained the funding. All co-authors approved the final version of the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To estimate the burden of diseases, it is important to consider patient-reported outcomes including Quality of Life (QoL). The aim of this study is to provide population-based reference data for the National Eye Institute 25-Item Visual Function Questionnaire (NEI VFQ-25), stratified by sex and age.The Gutenberg Health Study (GHS) is a population-based, prospective, observational cohort study in Germany, including 15,010 participants aged between 35 and 74. The baseline examination was conducted between 2007 and 2012. To overcome known shortcomings of the NEI VFQ-25, we calculated the previously proposed visual functioning scale and the socio-emotional scale based on Rasch-transformed person-level data. We present mean values, standard deviations and percentiles for age decades stratified by sex. We used a linear regression model to assess the influence of age, sex, socioeconomic status, distance-corrected visual acuity (better-seeing eye) and the absolute difference in distance-corrected visual acuity of both eyes on vision-related QoL.p < 0.001). For the LFSES score, we showed that the influence of sex was minor, and that age (per 5 years −0.22), visual acuity of the better eye (−1.65), and the difference between both eyes (−0.56) were associated with a lower score .NEI VFQ-25 data are available from 12,231 participants (82%). Both the long-form visual functioning scale (LFVFS) and the long-form socio-emotional scale (LFSES) showed a clear age dependency, with an average LFVFS score of 92.8 for men and 90.5 for women in the youngest age group and 85.7 and 83.4 in the oldest age group, and a LFSES score of 98.3 for men and 98.1 in women in the youngest and 94.7 and 94.5 in the oldest decade. The largest difference was observed between the youngest age group (35–44 years) and the 45–54 years group. Men tended to have slightly higher scores than women. In the multivariable linear regression analysis, age (per 5 years −0.42), female sex (−1.57), worse distance-corrected visual acuity of the better eye (per 0.1 increase in logMAR −2.92) and the difference between both eyes (per 0.1 increase in logMAR −0.87) were associated with a reduced LFVFS score  contains supplementary material, which is available to authorized users. The aim of this study was to provide vision-related quality of life (VRQoL) reference data from a large population-based sample for unidimensional scores based on Rasch-transformed NEI VFQ-25 data, and to assess the associations of age, sex, socio-economic status, and distance-corrected visual acuity with VRQoL. Subjective perception of diseases and their impact on daily life activities are important measurements to estimate the burden of diseases. Patient-reported outcomes are also gaining importance in the evaluation of therapeutic interventions. For example, the European Glaucoma Society Terminology and Guidelines state that the goal of glaucoma treatment is “to maintain the patient’s visual function and related quality of life” . There aThe Gutenberg Health Study (GHS) is a population-based, prospective, single-center cohort study at the medical center of the Johannes Gutenberg University Mainz in Germany . The pophttp://www.rand.org/health/surveys_tools/vfq.html, last accessed 2017–03-10). Subsequent studies showed that this approach would result in test scores violating unidimensionality and interval-level measurement, both important properties of an instrument measuring QoL [Vision-related QoL was assessed using the German version of the NEI VFQ-25 . The Gerring QoL , 26, 27.ring QoL , 29. RasAs previously described, refraction and distance-corrected visual acuity were measured in both eyes using a Humphrey Automated Refractor/Keratometer (HARK) 599 , starting with the right eye . Distanc2/kg. Cardiovascular disease was defined as history of ischemic heart disease, myocardial infarction, stroke, or peripheral arterial disease.Diabetes mellitus was defined by fulfilling one of the following criteria: diabetes mellitus diagnosed by a physician, known therapy , or HbA1c > =6.5%. Dyslipidemia was defined by a low-density lipoprotein (LDL) to high-density lipoprotein ratio (LDL/HDL) of >3.5, triglyceride levels after overnight fasting >150 mg/dL, lipid-lowering medication, or diagnosis by a physician. Hypertension was defined in the case of the use of antihypertensive medication, systolic blood pressure > 140 mmHg or diastolic blood pressure > 90 mmHg. Smoking was dichotomized into current smokers and non-smokers (including past smokers). Obesity was defined as a BMI > = 30 mThe socioeconomic status (SES) was based on income, education and occupation and was defined according to the SES-index as used within the German Health Update 2009 (GEDA), with a range from 3 to 21 (three indicates the lowest SES and 21 the highest SES) . Total yThe data management team performed quality controls for all data and checked for completeness and correctness by predefined algorithms and quality plausibility controls. Analyses were performed using SPSS version 23  and SAS software 9.4 . For descriptive analyses, we calculated the mean of the spherical equivalent and the intra-ocular pressure of both eyes for every participant. The distribution of NEI VFQ-25 scores was presented by calculating the mean, standard deviation, range and percentiles in steps of 5%, stratified by sex and decade of age. To assess a potential non-responder-bias, we compared the characteristics of participants who completed the questionnaire to participants with missing questionnaire information. We performed linear regression models to determine the association of the NEI VFQ-25 long-form visual functioning scale (LFVFS) and the long-form socio-emotional scale (LFSES) with age, sex, SES, and distance-corrected visual acuity. We additionally present distributions and associations of the short-form scales  to allow for comparison.Of the entire GHS population, 13,217 participants filled the NEI VFQ-25, and 12,231 (81.5%) participants completed the questionnaire without missing items necessary to calculate the proposed scores. Among participants with missing VRQoL information, comorbidities and risk factors such as diabetes mellitus (13.1% vs. 8.5%), hypertension (52.6% vs. 49.1%), dyslipidemia (48.7% vs. 43.3%), smoking (24.4% vs. 18.4%), obesity (29.7% vs. 24.2%) and cardiovascular disease (13.0% vs. 8.8%) were more frequent . All other factors also had a weak association with LFSES, compared to LFVFS: Per 5 year increase in age, the LFSES would decrease by 0.22. Per 0.1 logMAR increase in the better eye, the LFSES would decrease by 1.65. The difference of both eyes was associated with a decrease of 0.56 points per 0.1 logMAR increase.In the regression analysis, female participants had on average a 1.57 lower score in the LFVFS, assuming that the other factors  were constant  and the age group of 45–54 years. Men had higher scores than women. Lower score in LFVFS was also associated with lower visual acuity of the better-seeing eye as well as with the absolute difference in visual acuity between both eyes. These results are similar in all GHS participants as well as restricted to participants who reported having no eye diseases  , but didTo the best of our knowledge, this is the largest study to report NEI VFQ-25 scores for a population-based cohort and the first to report age- and sex-specific reference data for scales based on Rasch-transformed scores. We focused not only on the visual acuity of the better-seeing eye but also took into account both eyes. Due to the strong correlation between the fellow eyes, we decided not to simply include both eyes in the model but to additionally include the absolute difference in visual acuity between both eyes. In addition to the visual acuity of the better eye, the difference was associated with a smaller but still considerable reduction in the visual function scale score (−0.89 per 0.1 difference in logMAR). This underscores the importance of both eyes for vision-related QoL, which supports recent evidence that the worse-seeing eye might have a stronger impact on VRQoL than previously assumed , 38. TheThe strengths of our study are the standardized study design and quality control, the broad assessment of phenotype information, the large sample size, and the population-based sampling. Since the GHS baseline recruitment was limited to participants below the age of 75, we are not able to provide data on older age groups where the prevalence of eye diseases like AMD and glaucoma is significantly higher than in younger age groups. Individuals with visual impairment are likely to be underrepresented in the GHS cohort because of their lower likelihood to participate. Hirneiss et al. found a frequency of 17% of participants with an ophthalmological disease in their sample of a working-age population in Germany, which is twice the frequency of self-reported eye diseases in our sample . This do2 value of the regression models is 0.16 (LFVFS), meaning that only 16% of the visual functioning scale variation could be explained by the model.Even though the estimates from the linear regression are supported by the descriptive analyses (influence of sex and age), the results should be interpreted with care. The best RIn summary, we report age- and sex-specific VRQoL of two unidimensional scales based on Rasch-transformed NEI VFQ-25 questionnaire data from a large population-based German cohort that could be used as a reference for comparison in future studies. Furthermore, we show a clear age dependency for VRQoL, with lower scores at higher ages. The largest drop in VRQoL was observed between the age group of 35–44 years and the age group of 45–54 years. Worse VRQoL was associated with the visual acuity of the better-seeing eye and additionally with the difference between both eyes.


Multigravid women seemed to have the highest levels (p = 0.039). In infants, the level was highest in cord blood and decreased before stabilizing after 18 months (p<0.001). For children, a high level of sHLA-G was associated with malaria infection during the follow-up (p = 0.02) and low birth weight (p = 0.06). The mean level of sHLA-G during infancy was strongly correlated with the mother’s level during pregnancy (<0.001), and not only at delivery. Moreover, mothers with placental malaria infection had a higher probability of giving birth to a child with a high level of sHLA-g (p = 0.006). High sHLA-G levels during pregnancy might be associated with immune tolerance related to placental malaria. Further studies are needed but this study provides a first insight concerning the potential role of sHLA-G as a biomarker of weakness for newborns and infants.Human leukocyte antigen (HLA) G is a tolerogenic molecule involved in the maternal-fetal immune tolerance phenomenon. Its expression during some infectious diseases leading to immune evasion has been established. A first study conducted in Benin has shown that the production of soluble HLA-G (sHLA-G) during the first months of life is strongly correlated with the maternal level at delivery and associated with low birth weight and malaria. However sHLA-G measurements during pregnancy were not available for mothers and furthermore, to date the evolution of sHLA-G in pregnancy is not documented in African populations. To extend these previous findings, between January 2010 and June 2013, 400 pregnant women of a malaria preventive trial and their newborns were followed up in Benin until the age of 2 years. Soluble HLA-G was measured 3 times during pregnancy and repeatedly during the 2 years follow-up to explore how sHLA-G evolved and the factors associated. During pregnancy, plasma levels of sHLA-G remained stable and increased significantly at delivery ( Human leukocyte antigen-G (HLA-G) is a nonclassical HLA class I antigen that differs from the others HLA class I molecules in its limited polymorphism, its restricted tissue distribution and its particular expression . Alternain vitro fertilization ), 9.05% of them had a LBW, and 14 newborns were preterm. During the study, 12.16% of children developed a malaria infection between 6 and 24 months. There is no infection at birth in newborns.The general characteristics of the study population are presented in p<0.001).Blood was sampled in 85% of mothers for the three ANVs, and 98% were sampled at least twice. sHLA-G was described by month of pregnancy after grouping the first 3 months . The sHLp = 0.036) than primigravid women. The sHLA-G level seemed lower in women who took mefloquine for IPTp (p = 0.05) and those with placental malaria (p = 0.04). In contrast, there is no effect of age, peripheral malaria, foetus gender and LBW. In multivariate analysis, gravidity and IPTp remained significant , but the association of sHLA-G with placental malaria became marginally significant (p = 0.07).At the first ANV, univariate analysis showed that multigravid women had a higher sHLA-G level (p = 0.009)  had a higher sHLA-G level than the youngest (= 0.009) .p = 0.086 and p = 0.07, respectively)  .p<0.001)  .p = 0.02) (p = 0.06)  was not associated with the children’s sHLA-G level. In contrast, children infected by malaria at least once during the follow-up had a higher sHLA-G level ( = 0.02) . We iden = 0.06) .p<0.001) between each mother’s cross-sectional measurement and her child’s levels throughout the follow-up (p<0.001). Children born to a mother with very low profile had the lowest level, whereas children born to a mother with very high profile had the highest mean sHLA-G level.As described above, the sHLA-G level in children is correlated with their mother’s level. There were a strong correlation (ollow-up  and an ap<0.001). For an increase in the mother’s profile from “very low” to “low”, this OR was 3.05 (CI95 = [1.75–5.31]). The OR was 17.59 (CI95 = [9.54–32.41]) for an increase from “very low” to “very high”  occurred at different times for women . It could be that multigravid subjects produce more HLA-G and have a better aptitude for acceptance of semiallogenic foetal tissue. In other studies, a gravidity effect was not found , 16, butThe source of sHLA-G in newborns is not precisely known and documented to date. We hypothesize that this HLA-G could be produced by the organs of newborns himself such as thymus or umbilical cells  or immunP. falciparum could upregulate the HLA-G secretion by stimulating cytokines such as IL-10 and IFN-γ, which are well-known HLA-G inducers. HLA-G inhibits the function of T, NK, dendritic cells, neutrophils and B cells through direct interaction with ILT2 and/or ILT4 receptors [P. falciparum mediated by HLA-G may allow the parasite to escape the immune system and be responsible for higher susceptibility to infection. We have shown recently that high level of sHLA-G is associated with a higher probability to develop a malaria attack in the following weeks but also that the presence of malaria infection can increase sHLA-G that could lead to immune evasion [Malaria infection  in mothers has no effect on the sHLA-G level throughout pregnancy. This might be due to the substantial increase of sHLA-G in pregnancy, which could result in not detecting a malaria effect. It is noteworthy that pregnant women received two supervised doses of Intermittent Preventive Treatment against malaria, as recommended by WHO in all endemic areas, and that both the incidence of clinical malaria and parasitemia were low, for all IPT groups . Indepeneceptors , 43–47. eceptors , 9. Thereceptors . It has eceptors . Consequ evasion , 50. Ove evasion , 42 and The association between HLA-G and LBW shows that the evolution of sHLA-G during the first 2 years of life strongly differs between LBW children and others. LBW is correlated with infants’ morbidity and mortality , 21 and et al., even though the sHLA-G level in cord blood was lower than at term [Our results also confirmed the strong correlation between the mother’s sHLA-G levels at delivery and the cord blood level . Compara at term . However at term , 35, it  at term .Mother/child resemblance could have consequences from a public health point of view. If it is confirmed that high levels of sHLA-G are associated with LBW and a higher risk for malaria , the mother’s sHLA-G level during pregnancy could be a useful biomarker of frailty in children before delivery.It is accepted that children born from placental malaria are more susceptible to malaria during infancy, suggesting that this could stem from immune tolerance , 52, 53.


Additionally, the minor allele “A” of rs2853550 may also reduce the risk of AS in dominant (p = 0.025) and log-additive model (p = 0.024). Our results suggested that the polymorphisms of IL-1A and IL-1B are associated with the AS susceptibility in the Chinese Han population. Further studies are needed to characterize the functional sequences that cause AS.Ankylosing spondylitis (AS) is a complex and chronic inflammatory disease with a high heritage. Previous study has shown that IL-1A and IL-1B involved in inflammatory reaction. But little is known about single nucleotide polymorphisms (SNPs) of IL-1A and IL-1B associated with AS. We conducted a case-control study among 267 AS cases and 297 healthy controls from China. In the genetic model analysis, we found the “T” genotype of rs3783550 was associated with decreased AS risk in the dominant model ( Ankylosing spondylitis (AS) is a complex and chronic inflammatory disease with a high heritage. The overall prevalence of AS in Chinese is 0.2–0.54% . The conIL-1) cluster on chromosome 2q has emerged as a robust non-MHC susceptibility locus for AS in several groups study [IL-1 gene cluster and AS. Due to the IL-1 gene cluster also play a prominent role in inflammation and host defense against infection. The prototypic members of the IL-1 family gene cluster are the genes IL-1A, IL-1B, and IL-1RN [IL-1A and IL-1B were less reported [IL-1A and -B encode proinflammatory cytokines involved in host defense against infection [IL-1B was identified associated with AS in European ancestry [Although AS is also a complex multifactorial disease resulting from gene and environment interaction, the exact mechanism of AS is still unclear. As we all know, Genetic factors have an importance role in AS susceptibility and expression . HLA–B27ps study –7. Thosed IL-1RN , which wreported , 5, 8, 9nfection , 11. Rs1ancestry , suggestIL-1 genes were associated with AS in Chinese Han population, which is previously associated with AS in Europe population.Thus, in consideration of these circumstances, we sought to test whether p < 0.001).A total of 276 cases and 296 controls were enrolled in our study. The demographic characteristics of the study population are showed in Table p value screening, we excluded one SNP (rs2853550). The allelic frequency of SNPs in the controls group was similar to those of the HapMap Asian population. Through the χ2 test, we found that no SNP was significantly associated with AS risk.Table p = 0.044), and log-additive model . The minor allele “C” of rs3783546 was associated with a decreased AS risk under dominant model , and log-additive model . Additionally, the minor allele “A” of rs2853550 may also reduce the risk of AS, based on the dominant  and log-additive model . However, no statistically significant evidence suggested that the other polymorphisms tested were associated with AS risk.The minor allele of each SNP was assumed a risk allele compared to the wild-type allele. MAF in cases and controls are listed in Table IL-1A and IL-1B SNPs by haplotype analyses .Two blocks were detected in studied IL-1A and IL-1B SNPs and the risk of AS, rs3783550, rs3783546 and rs2853550 showed a protective effect on AS risk.In this Chinese Han population-based case–control study, we investigated the associations between the 12 SNPs and risk of AS. We found the associations between IL-1A were significance associated with a decreased risk of AS, which can act as a protect factor for AS. Although the individual polymorphism investigated showed no association with AS adjusted by age and sex, it is contribute to our hypothesis that substantially stronger haplotype associations with SNPs of IL-1A than with individual SNPs.The haplotype “TCG” of IL-1 gene family, the gene encoding their endogenous antagonist IL-lRa [IL1A and IL-1B may induce inflammation in AS through intracellular effects [IL1A and IL-1B are involved in the initiation of disease, but has a less significant role in established disease.IL-1A and IL-1B are one of the family of IL-1 and are proinflammatory cytokine that are mainly produced by activated macrophages, whose actions include augmentation of activation of T and B lymphocytes and monocyte/macrophages, and induce fibroblast proliferation leading to synovial pannus formation , which at IL-lRa . Randomit IL-lRa . This ma effects , it is aAS risk, only were associated with AS in different model in our study. That result might be due to differences among different regions of the world, which needs us to do further research to verify the results.A previous study found that rs3783550 was significantly associated with reduced AS risk in Canadian populations  and rheuDespite this study showed three SNPs  associated with AS susceptibility in the different model, some limitations should be considered. First, as the incidence of ankylosing spondylitis is low, so that the sample size of our study was relatively small. There may be some false positive results, but it has been able to explain the trend of IL-1A and IL-1B in patients with ankylosing spondylitis. In the future we will recruit more samples included in our future studies to verify our results. Second, the heterogeneity in living conditions was not evaluated in this study, which has contributed to the progress in elucidating the pathogenesis of AS.IL-1A), rs3783546 (IL-1A) and rs2853550 (IL-1B) and AS risk and between a new haplotype, “TCG”, of rs3783550, rs3783546 and rs2853550 and AS in Chinese Han population. Therefore, the results provide additional evidence for the relationships between genetic variants and AS susceptibility in the Hainan population. Moreover, a large number of genetic risk factors for AS should be investigated and validated to fully understand the pathogenesis of AS. Further studies will focus on the functional experiments based on the relevant genes on animal models, to investigate detailed mechanism involved.In conclusion, our study has described the association between rs3783550  using Fisher's exact test. The χ2 test was used to compare the distribution of marker alleles and genotypes in cases and controls [http://bioinfo.iconcologia.net. Values of OR and 95% CI were calculated as above.Akaike's InformationCriterion (AIC) and Bayesian InformationCriterion (BIC) were applied to choose the best-fit model for each SNP. Finally, we used the SHEsis software  to estimate the pairwise linkage disequilibrium (LD) and haplotype construction [We used Microsoft Excel and SPSS 18.0  to perform statistical analyses. In this study, all controls . Uncondicontrols . Associatruction .


Cats with pleural effusion represent common emergencies in small animal practice. The aim of this prospective study was to investigate the diagnostic ability of a point-of-care ELISA (POC-ELISA) for the measurement of N-terminal pro B-type natriuretic peptide (NT-proBNP) to differentiate cardiac from non-cardiac disease in cats with pleural effusion. The sample material for use of this rapid test was either plasma or diluted pleural effusion.Twenty cats with moderate to severe pleural effusion were prospectively recruited. The cats were grouped into two groups, with or without congestive heart failure , after complete work-up. Blood and effusion were collected in EDTA tubes. Plasma and pleural effusion supernatants were transferred into stabilizer tubes and frozen. POC-ELISA for NT-proBNP was performed with plasma and diluted effusion (1:1). Quantitative NT-proBNP measurement was performed in plasma and diluted and undiluted effusions.Six cats were assigned to the CHF group. Of the 14 cats in the N-CHF group, 6 had concurrent cardiac abnormalities that were not responsible for the effusion. For the detection of CHF, the test displayed respective sensitivities and specificities of 100% and 79% in plasma and 100% and 86% in diluted pleural fluid. Receiver operating characteristic (ROC) analysis for quantitative NT-proBNP measurement of plasma and diluted and undiluted pleural effusions displayed areas under the curve of 0.98, sensitivities of 100% and specificities of 86%. The optimum cut-off was calculated at 399 pmol/l in plasma and 229 pmol/l in the diluted effusion and 467 pmol/l in the undiluted effusion.POC-ELISA for NT-proBNP in both plasma and diluted pleural effusion was suitable to differentiate cardiac from non-cardiac causes of feline pleural effusion. According to our results, use of pleural effusion is feasible, but dilution of the effusion before measurement seems to improve specificity.The online version of this article (10.1186/s12917-017-1319-6) contains supplementary material, which is available to authorized users. Due to severe dyspnea, cats with pleural effusion are commonly presented to an emergency service. In addition to feline infectious peritonitis (FIP), pyothorax, neoplasia and idiopathic chylothorax, congestive heart failure (CHF) represents a common underlying disease , 2. ThorCardiac biomarkers have been used with increasing frequency to differentiate cardiac from non-cardiac dyspnea in small animal medicine. Studies have investigated the utility of NT-proBNP as a biomarker in cats –11. The First generation enzyme-linked-immunosorbent assay (ELISA) was used to measure the concentration of NT-proBNP in both plasma , 15 and Presence of relevant clinical signs leading to admission to the intensive care unit (ICU);Radiographic confirmation of moderate to severe pleural effusion (obliterated cardiac and diaphragmatic borders) ;Echocardiography request by the primary clinician.Cats with pleural effusion were consecutively included in the prospective study between March 2013 and April 2014. The following inclusion criteria had to be fulfilled:Radiography was performed either by the referring veterinarian or at presentation to the clinic. Initial echocardiography was performed either by a board-certified cardiologist or under his supervision by a cardiology resident or by a board-certified specialist in critical care to confirm the presence of pleural effusion and to classify the patient as being in congestive heart failure (CHF) or having a non-cardiogenic cause of pleural effusion (N-CHF). The group assignment (CHF versus N-CHF) was performed by a board-certified cardiologist according to full echocardiogram, history, physical examination and any additional work-up. Patients with CHF underwent complete echocardiographic examination (including Doppler-echocardiography and pulsed wave Tissue Doppler Imaging), ECG and Doppler-based blood pressure measurement. Underlying heart disease was classified according to the echocardiography results by a board-certified cardiologist. Cardiomyopathies were divided in 5 possible types according to published criteria : hypertrConcurrent cardiac abnormalities in patients with non-cardiac causes of pleural effusion (N-CHF group) were also recorded. Thoracocentesis was performed in all cats followed by pleural fluid analysis and cytological examination. In addition, a complete blood count and a biochemical panel were performed in all cats. After initial stabilization, the diagnostic work-up was performed based on the assignment to one of the two groups. In the N-CHF group, diagnostic investigation into the underlying non-cardiogenic disease was tailored to the individual patient. This included laboratory investigations  and diagnostic imaging modalities .Blood for NT-proBNP measurement was collected by venipuncture in EDTA-tubes. Pleural fluid samples were also placed in EDTA tubes and subsequently treated in the same way as blood samples. Sampling of blood and pleural effusion had a maximum time difference of 1 h. All samples in the EDTA tubes were centrifuged at 9000 x g for 1 min within 30 min of collection. EDTA plasma and EDTA pleural fluid supernatant were then placed in specific tubes containing a protease inhibitor mixtureA POC-ELISAIn addition, the following samples were shipped frozen to a commercial laboratoryument}2)  for statTo evaluate the effect of dilution of the effusion samples, the ratio between the NT-proBNP concentration in the paired undiluted and diluted samples was calculated for all samples with NT-proBNP concentrations within the detection limit of the assay.P values ≤ 0.05 were considered significant.Due to the small group size of the CHF group, non-Gaussian distribution was assumed. Descriptive statistics included frequencies for categorical variables and median and range for continuous variables. The data were compared between groups (CHF versus N-CHF) using the Mann-Whitney U test. Proportions were compared using Fisher’s exact test. The sensitivity and specificity and their 95% confidence interval (CI) for diagnosing CHF were calculated. Quantitative NT-proBNP values were graphically depicted as scatter plots of individual data points and were analyzed with receiver operator characteristic (ROC) analysis to determine the ability of NT-proBNP to diagnose CHF. The area under the curve (AUC) was used as a summary measure and quantification of diagnostic accuracy for NT-proBNP to predict CHF. The cut-off value was chosen based on the highest Youden index (Y = sensitivity + specificity - 1) . Statistn = 7), septic pyothorax (n = 3), hemothorax caused by chronic pleuritis (n = 1), feline infectious peritonitis (n = 1), pancreatitis (n = 1) and steatitis (n = 1). Six N-CHF patients had the following concurrent cardiac abnormalities: right atrial and ventricular dilation (n = 4), HCM (n = 1), and UCM (n = 1). A board-certified cardiologist judged that these changes were not severe enough to cause pleural effusion. In cats with CHF, the diagnoses were: HCM (n = 2), UCM (n = 2), DCM (n = 1), double chambered right ventricle (n = 1).Twenty cats were included in this study. Fourteen cats were classified as N-CHF and 6 as CHF. In the N-CHF patients, the following underlying diseases were diagnosed: neoplasia (p = 0.043).Patient data are shown in Table p = 0.32) different between the N-CHF  and CHF groups .In both groups, there was one patient each with heart murmur, gallop rhythm and arrhythmia. The proportion of cats with abnormal auscultation findings was not significantly  different between the N-CHF and CHF cats . The AUC was 0.98 (95% CI: 0.92–1.00), and the optimal cut-off of 229 pmol/l had a sensitivity of 100% (95% CI: 54.1% to 100%) and a specificity of 85.7% (95% CI: 57.2% to 98.2%). The use of 200 pmol/l (transition point of the POC test) as a cut-off for quantitative testing led to a sensitivity of 100% (95% CI: 54.1% to 100%) and a specificity of 78.6% (95% CI: 49.2% to 95.3%).When testing the diluted pleural effusion, 2/14 (14%) cats in the N-CHF group and 6/6 (100%) cats with CHF were positive on the POC test, which led to a sensitivity of 100% (95% CI: 54.1% to 100%) and a specificity of 85.7% (95% CI: 57.2% to 98.2%) for a diagnosis of CHF. Likewise, the median NT-proBNP concentration in diluted pleural effusion  different between the N-CHF  and CHF cats . The AUC was 0.98 (95% CI: 0.92–1.00), and the optimal cut-off of 399 pmol/l gave a sensitivity of 100% (95% CI: 54.1% to 100%) and a specificity of 85.7% (95% CI: 57.2% to 98.2%).Three out of 14 21%) cats in the N-CHF group and 6/6 (100%) cats with CHF were positive on the POC test when plasma samples were evaluated, which resulted in a sensitivity of 100% (95% CI: 54.1% to 100.0%) and a specificity of 78.6% (95% CI: 49.2% to 95.3%) for the diagnosis of CHF for the cause of pleural effusion. The quantitative values of plasma NT-proBNP  had quantitative test results higher than 200 pmol/l in both diluted pleural effusion and in the plasma  lower median NT-proBNP values than did cats in the CHF group . The AUC was 0.98 (95% CI: 0.92–1.0), and the cut-off of 467 pmol/l yielded a sensitivity of 100% (95% CI: 54.1% to 100%) and a specificity of 85.7% (95% CI: 57.2% to 98.2%). The use of 200 pmol/l as a cut-off value gave a sensitivity of 100% and a specificity of 64.3%.The quantitative NT-proBNP measurements were also performed with undiluted pleural effusion. Cats in the N-CHF group had significantly .In the present study, POC-ELISA for NT-proBNP was able to differentiate between cardiac and non-cardiac causes of pleural effusion in cats using either plasma or diluted pleural effusion samples. In addition, there was significant agreement between automated and optical evaluation as described earlier , which mOur study supported recent results reported with plasma samples  as the POur study design was similar to earlier studies using the first generation test , 15 and The second generation quantitative NT-proBNP assay had good performance in the present as well as in a previous study . The cutPOC-ELISA and quantitative testing were both suitable as screening tests due to the high sensitivity in our study and the earlier study . The limThe major limitation of the study was the small number of patients, especially in the CHF group. Blood pressure was not routinely measured in non-cardiac cats.Examination of the pleural fluid was not considered in the data interpretation but could aid in establishing the diagnosis, e.g., in cats with pyothorax.POC-ELISA in pleural effusion was only evaluated in diluted but not undiluted samples. Comparison of undiluted and diluted pleural effusion with the POC-ELISA in the same patient could have shown more clearly the effect of dilution on the specificity.We measured all of our samples on the POC-ELISA in a batch, and it would be better to test each patient immediately after sampling. This was not possible, as the test was not available at the beginning of patient recruitment.To avoid any influence of any freeze-thaw cycles, simultaneous measurements of quantitative NT-proBNP would have been ideal.In this small number of patients using plasma and diluted pleural effusion, NT-proBNP POC-ELISA was appropriate to differentiate between cardiac and non-cardiac underlying disease in cats with moderate to severe pleural effusion. Using pleural effusion, 1:1 dilution with 0.9% NaCl is feasible and has the potential to improve the diagnostic accuracy of this test with this sample material.


The prognostic stratification of patients with Idiopathic Dilated Cardiomyopathy (iDCM) is a difficult task. Here, we assessed the additive value of the evaluation of biomarkers of inflammasome activation and systemic inflammation for the long-term risk stratification of iDCM patients.We studied 156 ambulatory iDCM patients  35%, mean sodium 139 mEq/L, median BNP 189 pg/mL, median IL-1 beta (IL-1β) 1.08 pg/mL, median IL-6 1.7 pg/mL and median IL-10 2.7 pg/mL).During the follow-up period of 89.6 months, 35 patients (22%) died/underwent heart transplantation. Patients who died/underwent heart transplantation were more likely to be male, to be in NYHA class III, to have atrial fibrillation, to have lower LVEF and higher BNP levels. IL-1β, IL-6 and IL-10 levels did not differ significantly between the groups of patients with good or bad prognosis. IL-1β levels did not vary significantly among either the different NYHA classes or the quartiles of LVEF. In a multivariable model, however, IL-1β was a strong and independent predictor of all-cause mortality . Other factors associated with the outcome were: male gender, presence of atrial fibrillation and sodium concentration. The estimated time-dependent ROC curve of the multivariable model showed an AUC 0.74 (95% CI 0.65-0.86).Serum levels of IL-1β could be useful to predict the long-term outcome of patients with iDCM. Idiopathic Dilated Cardiomyopathy (iDCM) is the most frequent indication for cardiac transplantation . Being mPhysicians need to counsel patients about prognosis to enable informed decisions about medication and life expectancy. However, given the heterogeneity of the pathogenic mechanisms that lead to cardiac remodeling and heart failure, emerging mechanisms responsible for disease progression may help to identify and validate helpful biomarkers to prognostically stratify patients. In this regard, the sterile inflammatory response that occurs as a result of the activation of the inflammasome is an emerging mechanism responsible for promoting cardiac remodeling and heart failure (reviewed in ). ImportHowever, no literature data are available that evaluated the impact exerted by inflammasome activation on the long-term prognosis of iDCM patients. Therefore, we sought to assess the additive value of the evaluation of plasma biomarkers of inflammasome activation and systemic inflammation on top of traditional clinical variables for risk stratification of long-term mortality/heart transplantation in patients with heart failure affected by iDCM.Patients had a median age of 58 years, 77% were male, and only 21% were in NYHA class III (being the remaining 79% in NYHA class I-II). Table With regard to IL-1β, we observed significant negative correlations between this cytokine levels and both LV mass  and the presence of moderate-severe mitral valve regurgitation .Additionally, BNP plasma concentration positively correlated with that of IL-6 .Besides being positively correlated with BNP, IL-6 was significantly positively correlated with the NYHA III functional class , and both with the LVEDV index  and the LVESV index . As opposed, IL-6 levels were negatively correlated with LVEF .Finally, IL-10 concentrations did not correlate significantly with any of the variables that we evaluated.During the follow-up period , 35 patients (22%) died or underwent heart transplantation (31 deaths and 4 cardiac transplantations). Patients who died/underwent heart transplantation during the follow-up were more likely to be male, in NYHA class III, with atrial fibrillation and lower LV ejection fraction. The univariable analysis showed that these variables were even long-term predictors of outcome  were independently associated with long term mortality/cardiac transplantation the following variables: male gender, presence of atrial fibrillation, plasma sodium concentration and the transformed logarithmic squared version of IL-1β values Table . The estThis report represents the multicenter observational study with the largest cohort of real-life outpatients affected by heart failure secondary to iDCM in whom the long-term impact of inflammasome activation on mortality/need for cardiac transplantation has been described. The main finding of this study is that the prognostic information provided by the measurement of IL-1β plasma levels is independent of and beyond that provided by other relevant clinical factors, including the left ventricular ejection fraction as well as the degree of neurohumoral activation (sodium levels and plasma BNP).The progression of heart failure is complex and is driven by multiple biologic processes, such as neurohormonal activation and inflammation.As introduced, an accumulating body of literature demonstrates the primary role played by inflammasome activation in the evolution of heart disease towards cardiac remodeling and heart failure. A recent work by Luo and collaborators has recently measured inflammasome components in peripheral blood mononuclear cells and IL-1β in the plasma of 54 iDCM patients and 20 healthy controls. The study showed that NLRP3 and IL-1β mRNA levels are independent predictors of the necessity for rehospitalization in a six month follow-up .With regard to possible comorbidities that may influence IL-1β levels diabetes , myocardAlong with IL-1β, we investigated also whether IL-6 plasma levels are able to predict the prognosis of patients affected by iDCM. IL-6 is a pleiotropic cytokine that has been shown to exert both protective actions (e.g. favoring the resolution of the neutrophilic infiltrate) and to participate to disease pathogenesis (e.g. promoting the monocyte infiltration). Studies conducted on iDCM patients have shown increased production of IL-6 . HoweverIn addition to inflammatory processes, the complex pathways of neurohormonal activation are observed and their quantification through biomarkers might relay important information about heart failure. In our study, serum sodium levels were independently associated with the long-term outcome. In the setting of heart failure, hyponatremia reflects more severe activation of the renin–angiotensin–aldosterone and of the sympathetic nervous system as well as increased vasopressin release . Also, hThe present study focused primarily on the long-term mortality/need for cardiac transplantation of patients with iDCM. We accept as a limitation that we could not provide information on the time-course of plasma biomarker levels since the blood sample for biomarker analysis was drawn only at baseline.In conclusion, serum levels of IL-1β were able to prognostically stratify our cohort of iDCM patients. The precise cut-off of IL-1β associated with bad outcome in iDCM remains to be determined in larger, independent cohorts of patients.th, 2006 and January 31st, 2010 . The diagnosis of iDCM was made according to the World Health Organization criteria [A cohort of 156 patients with iDCM referred to Cardiology of Trieste and Monzino was prospectively enrolled between March 30criteria .Clinical and instrumental data were collected at the moment of enrolment and during each follow-up visit. Patients had schedule of visits at six, 12, 24 months and, subsequently, every two years or more frequently on the base of specific clinical needs.Blood samples were collected in ethylenediaminetetraacetic acid, immediately placed on ice, processed, and stored at -80°C until analysis was performed. The same blood sample was employed to test the concentrations of B-type natriuretic peptide (BNP), IL-1β, Interleukin 6 (IL-6 ) and Interleukin 10 (IL-10). We tested BNP also, since natriuretic peptides are currently the benchmark against which all biomarkers for prognosis of patients with heart failure must be measured.The quantitative determination of the BNP was measured from plasma samples obtained by centrifuging whole blood samples at 2,500g for 10′ at 4°C. Plasma levels of BNP were assayed using the competitive ELISA immunoassay RayBio (r) BNP Enzyme Immunoassay , according to the manufacturer's protocol. The minimum detectable concentration of BNP was: 1.66 pg/ml with an intra-assay coefficient of variation (CV) of < 10 % and an inter-assay CV of < 15 %.For the determination of plasma levels of IL-1 β, a high-sensitivity ELISA kit  has been used. The minimum detectable dose of Il-1β ranged from 0.023 to 0.140 pg/mL. The mean minimum detectable dose was 0,057 pg/mL. The concentration of IL-1β has been measured by subtracting the readings at 650nm from the readings at 450nm at the spectrophotometer.IL-6 High sensitivity sandwich ELISA  was used to measure IL-6 plasma levels. The minimum detectable dose was of 0.11 pg/mL while the kit's range was 0.2 - 10 pg/mL.Plasma levels of IL-10 were measured with The Quantikine Human IL-10 Immunoassay solid phase ELISA . This immunoassay has been shown to quantify recombinant human IL-10 and does not cross-react with viral IL-10. The optical density was determined at 450nm with wavelength correction at 570 nm by using a microplate reader. The sensitivity of the test of human IL-10 was less than 3.9 pg/mL.The main endpoint was all-cause mortality/urgent heart transplantation. For the objective of our study urgent heart transplantation was considered in status I, indicated in patients with refractory heart failure with necessity of inotropic treatment and/or mechanical support of the circulation.st, 2015 or the date of death or heart transplantation of the patient. This study conforms to the principles outlined in the Declaration of Helsinki and was approved by the institutional ethics committee. All patients provided written informed consent prior to enlistment in the Registry.Information regarding the end-points were obtained directly from the patient, from their family doctor or from the registers of death of the communes of residence. The end of the follow-up was set to December 31Characteristics of the study population are described using means ± s.d. or median and interquartile range for continuous variables, depending on the distribution's shape, and percentages for categorical variables. Data were tested for normal distribution using the Kolmogorov-Smirnov test. T-test or Mann-Whitney test, as appropriate, were used to compare continuous variables between two groups. For categorical variables, cross-tabulations were generated, and chi-square or Fisher exact test was used to compare distributions.To test the capacity of the biohumoral parameters to prognostically stratify iDCM patients, we analyzed death or necessity of cardiac transplantation as endpoint. Cox univariable analysis was performed and clinically relevant covariates as well as those with p < 0.1 were selected for multivariable analysis. Retention in the final stepwise model required the variable to be significant at p < 0.05 at multivariate analysis. Results are presented as hazard ratios (HR) and 95% confidence intervals (95% CI). The proportional hazards assumption of the Cox model was checked using the Therneau and Grambsch test.Since distributions of the biohumoral parameters showed a marked asymmetry, we performed a logarithmic transformation of their values and repeated the stepwise procedure using the log-transformed values. Log transformed data confirmed our findings on mortality/cardiac transplantation.Finally, the obtained score from the multivariable model was evaluated in terms of predictive accuracy by means of time-dependent ROC curve suitable for censored data . Analyse


Nowadays, there is a growing interest in compounds derived from plants as potential raw materials for drug development. One of the most studied compounds is beta-carotene (BC). Several clinical studies can be found investigating the cardiovascular effects of BC, however, all these results are controversial. There is an increasing body of evidence showing that besides the well-known antioxidant properties, under strong oxidative circumstances, BC could become prooxidant as well. In this study, we investigated the effects of long-term, low- and high-dose BC treatment in ischemic/reperfused (ISA/REP) hearts isolated from Zucker diabetic fatty (ZDF) rats. The animals were treated with various daily doses of BC for 4 weeks and then hearts were isolated and subjected to 30 min of global ischemia (ISA) followed by 120 min of reperfusion (REP). Blood glucose levels were measured before, after two weeks, and at the end of the treatment. In isolated hearts, the myocardial function was registered. At the end of the reperfusion period, the infarct size (IS) and heme oxygenase-1 (HO-1) expression were measured. The results showed that a low dose of BC treatment significantly improved postischemic recovery, which was reflected in a decreased IS. Interestingly, when BC was applied at high concentrations, the observed protective effects were lost. Although BC treatment increased HO-1 expression, we did not observe a better heart function and/or decreased IS in the high-dose-treated group. Glucose tolerance tests showed a concentration-independent decrease in blood glucose levels. Our results suggest that long-term, low-dose BC treatment could be effective in the treatment of type-2-diabetes and related cardiovascular diseases. A variety of materials of plant origin have been used for the treatment of different diseases. Nowadays, there is a growing interest in compounds derived from plants for the prevention and/or treatment of various disorders. Many plants contain flavonoids, anthocyanidins, proanthocyanidines, and carotenoids, which are all well-documented antioxidants and help in other biological activities ,3,4,5,6.In the past decades, there has been a significant increase in the number of patients diagnosed with type-2-diabetes (T2D), which is a multifactorial disease and one of the major risk factors for the development of cardiovascular diseases ,8,9. BesDepending on the circumstances and the concentration of BC, the agent could function as an antioxidant or a prooxidant . The obsp < 0.05); but interestingly, not from those fed with HD-BC (#p < 0.05) (p < 0.05) from rats receiving 30 mg/kg/day BC, but not 150 mg/kg/day of the agent (th HD-BC A. Furthe < 0.05) A. No sig < 0.05) B. Howeve < 0.05) B. Likewihe agent C. The treatment of animals with BC did not result in any changes in heart rate D. The evp < 0.05). This protective effect is abolished in hearts taken from animals treated with HD-BC.The effect of BC treatment on the extent of infarct size after ISA/REP is shown in p < 0.05) (#p < 0.05) or LD-BC-treated animals (LD-I/R) (&p < 0.05)  B. Moreov < 0.05) B.p < 0.005) in blood glucose level which was independent from the applied dose of BC  administered to the animals is very high, hence a normal human diet may contain three orders of magnitude less daily BC . Since t2+ produced by the increased HO-1 activity in response to ISA/REP injury may be a critical factor in the role of BC in ISA/REP-induced heart damage, which could act either as a protective antioxidant agent or behave as a prooxidant molecule and accelerate ISA/REP-related syndromes. This conclusion is speculative and will require further analysis. The ongoing characterization of the role of Fe2+ related to HO-1 activity is under investigation by our laboratory. Furthermore, our work demonstrated that BC possesses a hypoglycemic effect. The role of HO-1 in mediating the cardiovascular effects of BC and the mechanism behind the hypoglycemic action of the agent needs to be further elucidated.In conclusion, the main findings of the results show that increasing the concentration of BC will not provide an additional benefit in the diabetic ischemic/reperfused myocardium. Our results suggest that the level of FeThe experiments were accomplished using adult male obese ZDF rats  with a body weight range of 350–400 g. The animals were fed with special rodent chow (Purina #5008) according to the supplier instructions in order to induce programmed and consistent development of T2D. All animals received humane care in compliance with the “Principles of Laboratory Animal Care”  and the “Guide for the Care and Use of Laboratory Animals”. The maintenance and treatment of animals used in the present study were additionally approved by the Institutional Animal Care and Use Committee of the University of Debrecen, Debrecen, Hungary . The rats were housed in wire-bottomed cages (three rats per cage) throughout the study and were maintained on a 12:12-h light-dark cycle and provided with laboratory rodent chow pellets and water ad libitum.n = 10/group) and treated by gavage with the following agents: hydroxyethyl cellulose-water (1:4) vehicle control (C), LD-BC (30 mg/kg/day), and HD-BC (150 mg/kg/day) suspended in hydroxyethyl cellulose-water, respectively. It has to be mention that the applied doses exceed the levels from any natural sources of BC. Although rodents (rats and mice) readily convert BC to vitamin A, the bioavailability of the agent is low in case of absorption through the gut, requiring administration of BC at higher doses than physiologically required. The experiments were designed to partly offset this limitation. All-trans-BC was obtained from Sigma-Aldrich Kft. . The rats used in the present study were segregated into 3 groups .Following the 4-week treatment with the vehicle or BC, the animals were anesthetized with an intraperitoneal ketamine-xylazine injection (75/10 mg/kg), and intravenous heparin was given as an anticoagulant (1000 IU/kg). After thoracotomy, the hearts were excised and placed in ice-cold modified Krebs-Henseleit bicarbonate buffers. The isolated working rat heart model was used which has been previously described in detail . BrieflyAfter 10 min of working perfusion, 30 min of global ischemia was initiated by clamping the pulmonary inflow and the aortic outflow. At the end of the ischemic period, 120 min of reperfusion was started by unclamping the inflow and outflow lines. The first 10 min of reperfusion was conducted in Langendorff mode to avoid the development of fatal ventricular arrhythmias as described by Reference . The basw/v) solution of TTC in a phosphate buffer (pH 7.4) and the samples were stored at −70 °C for subsequent analysis. The frozen samples were sectioned, weighted, and blotted dry. The dried sections were scanned on an Epson J232D flat-bed scanner . The infarcted area (white coloration) and the risk area (entire scanned section) were measured using planimetry software . Estimates of the infarcted zone magnitude were subsequently obtained by multiplying the infarcted areas by the weight of each slice. The resulting numbers represent the weight of the risk zone and the infarcted zone. The infarct size was expressed as a percentage of the weight of the infarcted tissue and the weight of risk zone (whole heart).Estimations of the IS were conducted using the triphenyl tetrazolium chloride (TTC) staining method. Briefly, following each 30 min ischemia/120 min reperfusion period, the hearts were perfused with a 50 mL 1%  in an isolating buffer  and centrifuged at 2000 rpm at 4 °C for 10 min. The supernatants were transferred to fresh tubes and centrifuged at 10,000 rpm at 4 °C for 20 min, after which the resulting supernatant was used as the cytosolic fraction. The protein concentration was measured by ND-1000 Nano drop spectrophotometer with a BCA Protein Assay Kit . Thirty micrograms of protein in each sample were loaded in 10% polyacrylamide gel and resolved using SDS-PAGE electrophoresis and then transferred to a 0.45 µm pore size nitrocellulose membrane to concentrate the samples. After blocking the membranes with 7% nonfat dry milk in TBST, the membranes were incubated overnight with a primary antibody solution in 1% nonfat dry milk in TBST  at 4 °C. Then, the membranes were washed 3 times, each for 10 min, in TBST and incubated with a horseradish peroxidase-conjugated secondary antibody solution  containing 1% of nonfat dry milk in TBST, for two hours at room temperature. The membranes were treated with a Western blot Enhanced Chemiluminescent HRP substrate  to visualize the bands. After the Enhanced Chemiluminescent treatment, the membranes were exposed on X-ray films . The films were then digitalized by a flat-bed scanner  and analyzed using the ImageJ program and normalized the HO-1 band intensities to GAPDH.The content of the HO-1 protein in the myocardium was obtained by Western blot analysis. Approximately 300 mg of left ventricular myocardial tissue were homogenized on ice using a tissue homogenizer . The OGTT was performed as follows: after 12 h of fasting, the glucose levels were measured by sampling from the tail vein. Then, the rats were gavaged with 3 g/bwkg of glucose solution. The blood glucose levels were measured at the 30, 60, 120, and 180 min points after the administration of glucose.p < 0.05.Statistical analyses were performed using the GraphPad Prism 5 software . The data are expressed as the mean ± SEM. A one-way ANOVA followed by Newman-Keuls multiple comparison test was carried out for the data analysis. Any differences were considered significant at values of